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The zinc-finger transcription factor CASZ1 is required for differentiation of a distinct population of cardiomyocytes during development. However, expression of Casz1 mRNA is detected throughout the developing heart, suggesting the spatial regulation of CASZ1 occurs at the protein level. Relatively little is known about posttranscriptional regulation of Casz1 in the heart. We generated antibodies that specifically recognize CASZ1 in developing Xenopus embryos, and performed immunofluorescence analysis of CASZ1 during cardiac development. CASZ1 was detected throughout the developing myocardium. CASZ1 was restricted to terminally differentiated cardiomyocytes, and was down-regulated in cells that re-enter the cell cycle. We determined that CASZ1 expression correlated with terminal differentiation in cardiac muscle cells, skeletal muscle cells, and lymph-heart musculature. This study indicates that spatially distinct expression of CASZ1 protein may be due to posttranscriptional control of Casz1 mRNA during cardiac development. The results of this study provide insights into the role of Casz1 in cardiac function and in the differentiation of other cell types, including skeletal muscle and lymph heart.
Figure 1. Detection of CASZ1 in Xenopus embryos with anti-CASZ1 antibodies. A: Western blot of crude Xenopus lysates of uninjected and Casz1-V5 mRNA injected embryos (stage 12) demonstrates specificity of GP2381 anti-CASZ1 antibodies. Arrow denotes band corresponding to CASZ1-V5. B–M: Immunofluorescence of stage 40 Xenopus embryos (transverse sections) with three independent anti-CASZ1 antibodies. Both guinea pig antibodies (GP2381 and GP2384) and the single rabbit antibody (Rab701) recognize CASZ1 protein in cardiomyocyte nuclei throughout the heart. Dorsal is to the top. Anti-CASZ1 (B,F,J), anti-Tropomyosin (C,G,K), DAPI (D,H,L), and corresponding merge (E, I, M). Scale bar = 50 μm.Download figure to PowerPoint
Figure 2. CASZ1 protein is depleted in Casz1 morpholino (MO)-injected embryos. Maximum projections of z-stacks (50 μm total) through the linear heart tube (stage 34) of control (A–D) and MO injected embryos (E–H). Anterior to the left, dorsal to the top. Anti-CASZ1 (A, E), anti-Tropomyosin (B, F), DAPI (C,G), and corresponding merge (D,H). Scale bar = 50 μm.Download figure to PowerPoint
Figure 3. CASZ1 expression in the heart is restricted to the myocardium. A–D: Transverse section (10 μm) through the heart shows CASZ1 expression (A) is excluded from endocardium in the outflow tract (OFT) and atrioventricular canal (AVC) that is surrounded by Fibrillin (B). DAPI (C) and corresponding merge (D). V, ventricle, dorsal is to the topDownload figure to PowerPoint
Figure 4. CASZ1 is expressed throughout the myocardium. A–D: Maximum projections of z-stacks (50 μm total) through the looped heart (stage 43; ventral view). E–H: Transverse section (10 μm) of dorsal region of adult heartventricle. anti-CASZ1 (A,E), anti-Tropomyosin (B,F), DAPI (C,G), and corresponding merge (D,H). Scale bars = 50 μm.Download figure to PowerPoint
Figure 5. CASZ1 expression in the myocardium is correlated to the cell cycle. A–H: Transverse sections (10 μm, dorsal to the top) through the heart of a wild-type stage 40 embryo, with anti-CASZ1 (A,E), anti-phospho-histone H3 (B,F), merge (C,G), overlaid with anti-Tropomyosin staining (D,H). Arrows denote CASZ1− pHH3+ Tmy+ cardiomyocytes; arrowheads denote CASZ1+ pHH3+ Tmy+ cardiomyocytes. Scale bars = 50 μm. I: Quantification of cell counts of Tropomyosin+ cardiomyocyte nuclei from stage 40 embryos (n = 3; >500 cardiomyocytes scored per animal). Bars indicate standard deviation among animals scored.Download figure to PowerPoint
Figure 6. CASZ1 is expressed in all differentiated muscle in the embryo. A–E: Maximum projections of z-stacks (620.96 μm total) of stained stage 43 cardiac actin::gfp embryo, anterior half. F–J: Maximum projection of z-stacks (102.04 μm total) of magnified view of somites from embryo in A–E. K–O: Maximum projection of z-stacks (59.41 μm total) of magnified view of lymph heart from embryo in A–E. P–T: Maximum projection of z-stacks (180.29 μm total) of magnified view of heart from embryo in A–E. Anti-CASZ1 staining (A,F,K,P), anti-Tropomyosin (B,G,L,Q), anti-GFP (C,H,M,R), DAPI (D,I,N,S), and corresponding merge (E,J,O,T). Anterior to the left, dorsal to the top. Scale bars = 500 μm in E; 50 μm in J,O,T.Download figure to PowerPoint
Figure 7. CASZ1 expression in the lymph heart is also correlated to the cell cycle. A–H: Maximal projection of 100 μm of z-stacks through the somites (A–D) and magnified view of lymph heart in white box in panel D (E–H) of a stage 43 embryo, with anti-CASZ1 (A,E), anti-phospho-histone H3, pHH3 (B,F), merge (C,G), and overlaid with anti-Tropomyosin staining (D,H). Arrows denote CASZ1− pHH3+ lymph muscle nuclei. Arrowheads denote CASZ1+ pHH3+ lymph muscle nuclei.Download figure to PowerPoint
Figure 8. CASZ1 is expressed in differentiated neural tissue during development. A–C: Expression of CASZ1 in the dorsal portions of anterior neural tube. D–K: CASZ1 is present (D,H) in both eyes of stage 42 embryos and colocalizes (G,K) to DAPI-stained nuclei (F,J) of rod photoreceptors marked by Gαtransducin (E,I). Panels H–K are magnified views of eyes in D–G.Download figure to PowerPoint
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